Value of altered methylation patterns of genes RANBP3, LCP2 and GRAP2 in cfDNA in breast cancer diagnosis.

Background: The purpose of this study was to investigate the potential of plasma cfDNA methylation patterns in reflecting tumour methylation changes, focusing on three candidate sites, cg02469161, cg11528914, and cg20131654. These sites were selected for verification, with a particular emphasis on their association with breast cancer. Methods: We conducted a comprehensive analysis of 850k whole-methylation sequencing data to identify potential markers for breast cancer detection. Subsequently, we investigated the methylation status of the genes Ran-binding protein 3 (RANBP3), Lymphocyte cytoplasmic protein 2 (LCP2), and GRB2 related adaptor protein 2 (GRAP2), situated at the specified sites, using cancer and canceradjacent tissues from 17 breast cancer patients. We also examined the methylation patterns in different molecular subtypes and pathological grades of breast cancer. Additionally, we compared the methylation levels of these genes in plasma cfDNA to their performance in tissues. Results: Our analysis revealed that RANBP3, LCP2, and GRAP2 genes exhibited significant methylation differences between cancer and cancer-adjacent tissues. In breast cancer, these genes displayed diagnostic efficiencies of 91.0%, 90.6%, and 92.2%, respectively. Notably, RANBP3 showed a tendency towards lower methylation in HR+ breast cancer, and LCP2 methylation was correlated with tumour malignancy. Importantly, the methylation levels of these three genes in plasma cfDNA closely mirrored their tissue counterparts, with diagnostic efficiencies of 83.3%, 83.9%, and 77.6% for RANBP3, LCP2, and GRAP2, respectively. Conclusions: Our findings propose that the genes RANBP3, LCP2, and GRAP2, located at the identified methylation sites, hold significant potential as molecular markers in blood for the supplementary diagnosis of breast cancer. This study lays the groundwork for a more in-depth investigation into the changes in gene methylation patterns in circulating free DNA (cfDNA) for the early detection not only of breast cancer but also for various other types of cancer.

Methylation-Specific Electrophoresis.

Methylation of CpG sites in the promoter region of genomic DNA is an important epigenetic modification that plays a critical role in gene regulation, particularly in gene silencing. Epigenetic abnormalities, along with genetic alterations, are implicated in carcinogenesis and cancer progression. Numerous studies have investigated the role of epigenetics in cancer using various tools to assess DNA methylation. However, conventional analysis methods for DNA methylation require a large amount of DNA but lack higher sensitivity, making them unsuitable for analysis of samples with high heterogeneity, such as tumor tissues. In this study, we introduce a novel electrophoresis method named "methylation-specific electrophoresis (MSE)," which utilizes a denaturing gradient acrylamide gel. We demonstrate the applicability of the MSE method for DNA methylation analysis of the mucin gene as an example.

Estimating genome-wide DNA methylation heterogeneity with methylation patterns.

BACKGROUND: In a heterogeneous population of cells, individual cells can behave differently and respond variably to the environment. This cellular diversity can be assessed by measuring DNA methylation patterns. The loci with variable methylation patterns are informative of cellular heterogeneity and may serve as biomarkers of diseases and developmental progression. Cell-to-cell methylation heterogeneity can be evaluated through single-cell methylomes or computational techniques for pooled cells. However, the feasibility and performance of these approaches to precisely estimate methylation heterogeneity require further assessment. RESULTS: Here, we proposed model-based methods adopted from a mathematical framework originally from biodiversity, to estimate genome-wide DNA methylation heterogeneity. We evaluated the performance of our models and the existing methods with feature comparison, and tested on both synthetic datasets and real data. Overall, our methods have demonstrated advantages over others because of their better correlation with the actual heterogeneity. We also demonstrated that methylation heterogeneity offers an additional layer of biological information distinct from the conventional methylation level. In the case studies, we showed that distinct profiles of methylation heterogeneity in CG and non-CG methylation can predict the regulatory roles between genomic elements in Arabidopsis. This opens up a new direction for plant epigenomics. Finally, we demonstrated that our score might be able to identify loci in human cancer samples as putative biomarkers for early cancer detection. CONCLUSIONS: We adopted the mathematical framework from biodiversity into three model-based methods for analyzing genome-wide DNA methylation heterogeneity to monitor cellular heterogeneity. Our methods, namely MeH, have been implemented, evaluated with existing methods, and are open to the research community.

Methylation Dynamics on 5'-UTR of DAT1 Gene as a Bio-Marker to Recognize Therapy Success in ADHD Children.

Attention-deficit/hyperactivity disorder (ADHD), a neuropsychiatric condition characterized by inattention, hyperactivity, and impulsivity, afflicts 5% of children worldwide. Each ADHD patient presents with individual cognitive and motivational peculiarities. Furthermore, choice of appropriate therapy is still up to clinicians, who express somewhat qualitative advice on whether a child is being successfully cured or not: it would be more appropriate to use an objective biomarker to indicate whether a treatment led to benefits or not. The aim of our work is to search for such clinical biomarkers. We recruited 60 ADHD kids; psychopathological scales were administered at recruitment and after six weeks of therapy. Out of such a cohort of ADHD children, we rigorously extracted two specific subgroups; regardless of the initial severity of their disease, we compared those who obtained the largest improvement (ΔCGAS > 5) vs. those who were still characterized by a severe condition (CGAS < 40). After such a therapy, methylation levels of DNA extracted from buccal swabs were measured in the 5'-UTR of the DAT1 gene. CpGs 3 and 5 displayed, in relation to the other CpGs, a particular symmetrical pattern; for "improving" ADHD children, they were methylated together with CpG 2 and CpG 6; instead, for "severe" ADHD children, they accompanied a methylated CpG 1. These specific patterns of methylation could be used as objective molecular biomarkers of successful cures, establishing if a certain therapy is akin to a given patient (personalized medicine). Present data support the use of post-therapy molecular data obtained with non-invasive techniques.

Transcriptome-wide profiling of mRNA N6-methyladenosine modification in rice panicles and flag leaves.

N6-methyladenosine (m6A) modification is essential for plant growth and development. Exploring m6A methylation patterns in rice tissues is fundamental to understanding the regulatory effects of this modification. Here, we profiled the transcriptome-wide m6A landscapes of rice panicles at the booting stage (PB) and flowering stage (PF), and of flag leaves at the flowering stage (LF). The global m6A level differed significantly among the three tissues and was closely associated with the expression of writer and eraser genes. The methylated gene ratio was higher in the flag leaves than in the panicles. Compared with commonly methylated genes, tissue-specific methylated genes showed lower levels of both m6A modification and expression, and a preference for m6A deposition in the coding sequence region. The m6A profiles of the two organs had more distinct differences than the profiles of the same organ at different stages. A negative correlation between m6A levels and gene expression was observed in PF vs. PB but not in PF vs. LF, indicting the complicated regulatory effect of m6A on gene expression. The distinct expression patterns of m6A reader genes in different tissues indicate that readers may affect gene stability through binding. Overall, our findings demonstrated that m6A modification influences tissue function by regulating gene expression. Our findings provide valuable insights on the regulation and biological functions of m6A modifications in rice.

Methylome and transcriptome data integration reveals potential roles of DNA methylation and candidate biomarkers of cow Streptococcus uberis subclinical mastitis.

BACKGROUND: Mastitis caused by different pathogens including Streptococcus uberis (S. uberis) is responsible for huge economic losses to the dairy industry. In order to investigate the potential genetic and epigenetic regulatory mechanisms of subclinical mastitis due to S. uberis, the DNA methylome (whole genome DNA methylation sequencing) and transcriptome (RNA sequencing) of milk somatic cells from cows with naturally occurring S. uberis subclinical mastitis and healthy control cows (n = 3/group) were studied. RESULTS: Globally, the DNA methylation levels of CpG sites were low in the promoters and first exons but high in inner exons and introns. The DNA methylation levels at the promoter, first exon and first intron regions were negatively correlated with the expression level of genes at a whole-genome-wide scale. In general, DNA methylation level was lower in S. uberis-positive group (SUG) than in the control group (CTG). A total of 174,342 differentially methylated cytosines (DMCs) (FDR < 0.05) were identified between SUG and CTG, including 132,237, 7412 and 34,693 DMCs in the context of CpG, CHG and CHH (H = A or T or C), respectively. Besides, 101,612 methylation haplotype blocks (MHBs) were identified, including 451 MHBs that were significantly different (dMHB) between the two groups. A total of 2130 differentially expressed (DE) genes (1378 with up-regulated and 752 with down-regulated expression) were found in SUG. Integration of methylome and transcriptome data with MethGET program revealed 1623 genes with significant changes in their methylation levels and/or gene expression changes (MetGDE genes, MethGET P-value < 0.001). Functional enrichment of genes harboring ≥ 15 DMCs, DE genes and MetGDE genes suggest significant involvement of DNA methylation changes in the regulation of the host immune response to S. uberis infection, especially cytokine activities. Furthermore, discriminant correlation analysis with DIABLO method identified 26 candidate biomarkers, including 6 DE genes, 15 CpG-DMCs and 5 dMHBs that discriminated between SUG and CTG. CONCLUSION: The integration of methylome and transcriptome of milk somatic cells suggests the possible involvement of DNA methylation changes in the regulation of the host immune response to subclinical mastitis due to S. uberis. The presented genetic and epigenetic biomarkers could contribute to the design of management strategies of subclinical mastitis and breeding for mastitis resistance.

Competitive binding of TET1 and DNMT3A/B cooperates the DNA methylation pattern in human embryonic stem cells.

DNMT3A/B and TET1 play indispensable roles in regulating DNA methylation that undergoes extensive reprogramming during mammalian embryogenesis. Yet the competitive and cooperative relationships between TET1 and DNMT3A/B remain largely unknown in the human embryonic stem cells. Here, we revealed that the main DNA-binding domain of TET1 contains more positive charges by using charge reduction of amino acid alphabet, followed by DNMT3A and DNMT3B. The genome-wide binding profiles showed that TET1 prefers binding to the proximal promoters and CpG islands compared with DNMT3A/B. Moreover, the binding regions of these three transcription factors can be divided into specific and co-binding regions. And a stronger inhibitory effect of DNMT3A on TET1 demethylation was observed in co-binding regions. Furthermore, we integrated TET1 knockout data to further discuss the competitive binding patterns of TET1 and DNMT3A/B. The lack of TET1 increased the occupation of DNMT3A/B at the specific binding regions of TET1 causing focal hypermethylation. The knockout of TET1 was also accompanied by a reduction of DNMT3A/B binding in the co-binding regions, further confirming the cooperative binding function between TET1 and DNMT3A/B. In conclusion, our studies found that the competitive binding of TET1 and DNMT3A/B cooperatively shapes the global DNA methylation pattern in human embryonic stem cells.

The impact of NRG1 expressions and methylation on multifactorial Hirschsprung disease.

BACKGROUND: Hirschsprung disease (HSCR) is a complex genetic disorder characterized by the lack of ganglion cells in the intestines. A current study showed that the NRG1 rare variant frequency in Indonesian patients with HSCR is only 0.9%. Here, we investigated the impact of NRG1 expressions and methylation patterns on the pathogenesis of HSCR. METHODS: This cross-sectional study determined NRG1 type I (HRGα, HRGß1, HRGß2, HRGß3, HRGγ, and NDF43 isoforms), type II and type III expressions in both ganglionic and aganglionic colons of 20 patients with HSCR and 10 control colons by real-time polymerase chain reaction (qPCR). For methylation studies, we treated the extracted gDNA from 16 HSCR patients' and 17 control colons with sodium bisulfate and analyzed the methylation pattern of NRG1 exon 1 with methylation-specific PCR. The samples were collected and analyzed at our institution from December 2018 to December 2020. RESULTS: NRG1 types I, II and III expressions were upregulated (17.2-, 3.2-, and 7.2-fold, respectively) in the ganglionic colons compared with control colons (type I: 13.32 ± 1.65 vs. 17.42 ± 1.51, p < 0.01; type II: 13.73 ± 2.02 vs. 16.29 ± 2.19, p < 0.01; type III: 13.47 ± 3.01 vs. 16.32 ± 2.58, p = 0.03; respectively); while only type I (7.7-fold) and HRGß1/HRGß2 (3.3-fold) isoforms were significantly upregulated in the aganglionic colons compared to the controls (type I: 14.47 ± 1.66 vs. 17.42 ± 1.51, p < 0.01; HRGß1/HRGß2: 13.62 ± 3.42 vs 14.75 ± 1.26, p = 0.01). Moreover, the frequency of partially methylated NRG1 was higher in the ganglionic (81%) and aganglionic (75%) colons than in the controls (59%). CONCLUSIONS: Our study provides further insights into the aberrant NRG1 expression in the colons of patients with HSCR, both ganglionic and aganglionic bowel, which might contribute to the development of HSCR, particularly in Indonesia. Furthermore, these aberrant NRG1 expressions might be associated with its methylation pattern.

Genome-wide DNA Methylation Differences in Nonfunctioning Pituitary Adenomas With and Without Postsurgical Progression.

CONTEXT: Tumor progression in surgically treated patients with nonfunctioning pituitary adenomas (NFPAs) is associated with excess mortality. Reliable biomarkers allowing early identification of tumor progression are missing. OBJECTIVE: To explore DNA methylation patterns associated with tumor progression in NFPA patients. METHODS: This case-controlled exploratory trial at a university hospital studied patients who underwent surgery for NFPA that had immunohistochemical characteristics of a gonadotropinoma. Cases included patients requiring reintervention due to tumor progression (reintervention group, n = 26) and controls who had a postoperative residual tumor without tumor progression for at least 5 years (radiologically stable group, n = 17). Genome-wide methylation data from each tumor sample were analyzed using the Infinium MethylationEPIC BeadChip platform. RESULTS: The analysis showed that 605 CpG positions were significantly differently methylated (differently methylated positions, DMPs) between the patient groups (false discovery rate adjusted P value < 0.05, beta value > 0.2), mapping to 389 genes. The largest number of DMPs were detected in the genes NUP93 and LGALS1. The 3 hypomethylated DMPs and the 3 hypermethylated DMPs with the lowest P values were all significantly (P < 0.05) and individually associated with reintervention-free survival. One of the hypermethylated DMPs with the lowest P value was located in the gene GABRA1. CONCLUSION: In this exploratory study, DNA methylation patterns in NFPA patients were associated with postoperative tumor progression requiring reintervention. The DMPs included genes that have been previously associated with tumor development. Our study is a step toward finding epigenetic signatures to predict tumor progression in patients with NFPA.

Olive Oil Improves While Trans Fatty Acids Further Aggravate the Hypomethylation of LINE-1 Retrotransposon DNA in an Environmental Carcinogen Model.

DNA methylation is an epigenetic mechanism that is crucial for mammalian development and genomic stability. Aberrant DNA methylation changes have been detected not only in malignant tumor tissues; the decrease of global DNA methylation levels is also characteristic for aging. The consumption of extra virgin olive oil (EVOO) as part of a balanced diet shows preventive effects against age-related diseases and cancer. On the other hand, consuming trans fatty acids (TFA) increases the risk of cardiovascular diseases as well as cancer. The aim of the study was to investigate the LINE-1 retrotransposon (L1-RTP) DNA methylation pattern in liver, kidney, and spleen of mice as a marker of genetic instability. For that, mice were fed with EVOO or TFA and were pretreated with environmental carcinogen 7,12-dimethylbenz[a]anthracene (DMBA)-a harmful substance known to cause L1-RTP DNA hypomethylation. Our results show that DMBA and its combination with TFA caused significant L1-RTP DNA hypomethylation compared to the control group via inhibition of DNA methyltransferase (DNMT) enzymes. EVOO had the opposite effect by significantly decreasing DMBA and DMBA + TFA-induced hypomethylation, thereby counteracting their effects.

DNA Methylation Patterns Differ between Free-Living Rhizobium leguminosarum RCAM1026 and Bacteroids Formed in Symbiosis with Pea (Pisum sativum L.).

Rhizobium leguminosarum (Rl) is a common name for several genospecies of rhizobia able to form nitrogen-fixing nodules on the roots of pea (Pisum sativum L.) while undergoing terminal differentiation into a symbiotic form called bacteroids. In this work, we used Oxford Nanopore sequencing to analyze the genome methylation states of the free-living and differentiated forms of the Rl strain RCAM1026. The complete genome was assembled; no significant genome rearrangements between the cell forms were observed, but the relative abundances of replicons were different. GANTC, GGCGCC, and GATC methylated motifs were found in the genome, along with genes encoding methyltransferases with matching predicted target motifs. The GGCGCC motif was completely methylated in both states, with two restriction-modification clusters on different replicons enforcing this specific pattern of methylation. Methylation patterns for the GANTC and GATC motifs differed significantly depending on the cell state, which indicates their possible connection to the regulation of symbiotic differentiation. Further investigation into the differences of methylation patterns in the bacterial genomes coupled with gene expression analysis is needed to elucidate the function of bacterial epigenetic regulation in nitrogen-fixing symbiosis.

Whole-Genome Methylation Analysis Revealed ART-Specific DNA Methylation Pattern of Neuro- and Immune-System Pathways in Chinese Human Neonates.

The DNA methylation of human offspring can change due to the use of assisted reproductive technology (ART). In order to find the differentially methylated regions (DMRs) in ART newborns, cord blood maternal cell contamination and parent DNA methylation background, which will add noise to the real difference, must be removed. We analyzed newborns' heel blood from six families to identify the DMRs between ART and natural pregnancy newborns, and the genetic model of methylation was explored, meanwhile we analyzed 32 samples of umbilical cord blood of infants born with ART and those of normal pregnancy to confirm which differences are consistent with cord blood data. The DNA methylation level was lower in ART-assisted offspring at the whole genome-wide level. Differentially methylated sites, DMRs, and cord blood differentially expressed genes were enriched in the important pathways of the immune system and nervous system, the genetic patterns of DNA methylation could be changed in the ART group. A total of three imprinted genes and 28 housekeeping genes which were involved in the nervous and immune systems were significant different between the two groups, six of them were detected both in heel blood and cord blood. We concluded that there is an ART-specific DNA methylation pattern involved in neuro- and immune-system pathways of human ART neonates, providing an epigenetic basis for the potential long-term health risks in ART-conceived neonates.

Promoter Methylation Status and Expression Levels of RASSF1A Gene in Different Phases of Acute Lymphoblastic Leukemia (ALL).

Background: Although the precise pathogenesis of acute lymphoblastic leukemia (ALL) remains unclear, studying gene-regulating mechanisms during ALL pathogeneses may shed light on the underlying mechanisms driving malignant behavior. There is some evidence showing the promoter hypermethylation and silencing of RASSF1A tumor suppressor gene in ALL cells; however, there is a lack of evidence for whether the gene indeed alters during different phases of ALL or in response to therapy. Thus, the current study aimed to clarify this issue using groups of adult ALL patients who have been scarcely investigated regarding expression levels and promoter methylation status. Materials and Methods: In this case/control study, the expression levels and methylation status of the gene promoter was evaluated using quantitative real-time PCR and methylation-specific PCR (MSP), respectively in adults with ALL. The study included peripheral blood of patients with newly diagnosed ALL (n=10), complete remission (CR) (n=10), or relapse (n=10), and 10 control samples from healthy individuals. Results: MSP results revealed an unmethylated status for almost all patients and control samples, except a case with relapsing ALL, which showed a hemimethylated pattern. RASSF1A also showed no difference in terms of gene expression in the patients compared with the control group (p>0.05). Conclusion: The results revealed an up-regulation of RASSF1A tumor suppressor in adult ALL patients experiencing CR, suggesting this to be a marker of therapy response. However, further investigations using more sensitive methylation detecting tools with larger sample sizes may better clarify the involvement of the promoter methylation of RASSF1A in these patients.

Clinical Response to Traditional Chinese Herbs Containing Realgar (As2S2) is Related to DNA Methylation Patterns in Bone Marrow DNA from Patients with Myelodysplastic Syndrome with Multilineage Dysplasia.

PURPOSE: DNA methylation is known to play an important role in myelodysplastic syndrome (MDS). We previously showed that Chinese herbs (CHs) containing realgar (As2S2) were effective at treating MDS with multilineage dysplasia (MDS-MLD). We tested whether the response to CH treatment was related to changes in DNA methylation in MDS-MLD. PATIENTS AND METHODS: First, the Illumina methylation 850K array BeadChip assay was used to assess the pretreatment methylation status in bone marrow cells from eight MDS-MLD patients and 3 healthy donors. The eight MDS-MLD patients were then treated with CHs for six months, the arsenic concentration was measured following treatment. The patients were subsequently divided into "effective" and "ineffective" treatment response groups and the DNA methylation patterns of the two groups were compared. Finally, the BeadChip data were validated by pyrosequencing. RESULTS: Five of the eight MDS-MLD patients showed hematological improvement (effective-treatment group), while three showed disease progression (ineffective-treatment group) (positive response rate: 62.5%). The arsenic concentrations in the patients ranged from 26.60 to 64.16 µg/L (median 48.4 µg/L) and were not significantly different between the two groups (p = 0.27). Compared with the healthy controls, three genes were hypomethylated and 110 were hypermethylated in the ineffective-treatment group. However, in the group showing hematological improvement, 102 genes were markedly hypomethylated and 87 hypermethylated. The effective-treatment group had a higher proportion of hypomethylated sites than the ineffective-treatment group (53.9% vs 2.6%, respectively; chi-square test) (p < 0.0001). Two hypermethylated and two hypomethylated genes were selected for validation by pyrosequencing (all p < 0.05). CONCLUSION: MDS-MLD patients may present different DNA methylation subtypes. CHs containing realgar may be effective for treating MDS-MLD patients with the hypomethylation subtype.

FMR1 expression in human granulosa cells and variable ovarian response: control by epigenetic mechanisms.

In humans, FMR1 (fragile X mental retardation 1) is strongly expressed in granulosa cells (GCs) of the female germline and apparently controls efficiency of folliculogenesis. Major control mechanism(s) of the gene transcription rate seem to be based on the rate of CpG-methylation along the CpG island promoter. Conducting CpG-methylation-specific bisulfite-treated PCR assays and subsequent sequence analyses of both gene alleles, revealed three variably methylated CpG domains (FMR1-VMR (variably methylated region) 1, -2, -3) and one completely unmethylated CpG-region (FMR1-UMR) in this extended FMR1-promoter-region. FMR1-UMR in the core promoter was exclusively present only in female GCs, suggesting expression from both gene alleles, i.e., escaping the female-specific X-inactivation mechanism for the second gene allele. Screening for putative target sites of transcription factors binding with CpG methylation dependence, we identified a target site for the transcriptional activator E2F1 in FMR1-VMR3. Using specific electrophoretic mobility shift assays, we found E2F1 binding efficiency to be dependent on CpG-site methylation in its target sequence. Comparative analysis of these CpGs revealed that CpG 94-methylation in primary GCs of women with normal and reduced efficiency of folliculogenesis statistically significant differences. We therefore conclude that E2F1 binding to FMR1-VMR3 in human GCs is part of an epigenetic mechanism regulating the efficiency of human folliculogenesis. Our data indicate that epigenetic mechanisms may control GC FMR1-expression rates.

Differential Methylation Pattern of Schizophrenia Candidate Genes in Tetrahydrocannabinol-Consuming Treatment-Resistant Schizophrenic Patients Compared to Non-Consumer Patients and Healthy Controls.

BACKGROUND: Patients suffering from schizophrenic psychosis show reduced synaptic connectivity compared to healthy individuals. Furthermore, the use of cannabis often precedes the onset of schizophrenic psychosis. Therefore, we investigated whether consumption of cannabis has an impact on the methylation pattern of schizophrenia candidate genes concerned with the development and preservation of synapses and synaptic function. METHODS: Fifty blood samples of outpatients affected by treatment-resistant schizophrenic psychosis were collected in the outpatient department of Ch Ste Anne/INSERM (Paris, France). Extracted DNA was sent to the LMN/MHH (Hanover, Germany) where DNA samples were bisulfite converted. The methylation patterns of the promoter region of neuregulin 1 (NRG1), neurexin (NRXN1), disrupted in schizophrenia 1 (DISC1), and microtubule-associated-protein tau (MAPT) were then analysed by sequencing according to Sanger. RESULTS: In NRXN1 the group of non-consumer patients showed a methylation rate slightly lower than controls. In patients with preliminary use of tetrahydrocannabinol (THC) the NRXN1 promoter turned out to be methylated almost two times higher than in non-consumer patients. In MAPT, non-consumer patients showed a significant lower mean methylation rate in comparison to controls. In THC-consuming patients the difference compared with controls became less. NRG1 and DISC1 showed no significant differences between groups, whereas DISC1 appeared to be not methylated at all. CONCLUSION: In MAPT and NRXN1 mean methylation rates were lower in non-consumer patients compared with controls, which seems to be a compensatory mechanism. With consumption of THC, mean methylation rates were increased: in the case of MAPT compared with controls, and in NRXN1 even significantly beyond that. Methylation of NRG1 and DISC1 seems not to be affected by the psychiatric disorder or by consumption of THC.

Molecular basis for histone H3 "K4me3-K9me3/2" methylation pattern readout by Spindlin1.

Histone recognition by "reader" modules serves as a fundamental mechanism in epigenetic regulation. Previous studies have shown that Spindlin1 is a reader of histone H3K4me3 as well as "K4me3-R8me2a" and promotes transcription of rDNA or Wnt/TCF4 target genes. Here we show that Spindlin1 also acts as a potent reader of histone H3 "K4me3-K9me3/2" bivalent methylation pattern. Calorimetric titration revealed a binding affinity of 16 nm between Spindlin1 and H3 "K4me3-K9me3" peptide, which is one to three orders of magnitude stronger than most other histone readout events at peptide level. Structural studies revealed concurrent recognition of H3K4me3 and H3K9me3/2 by aromatic pockets 2 and 1 of Spindlin1, respectively. Epigenomic profiling studies showed that Spindlin1 colocalizes with both H3K4me3 and H3K9me3 peaks in a subset of genes enriched in biological processes of transcription and its regulation. Moreover, the distribution of Spindlin1 peaks is primarily associated with H3K4me3 but not H3K9me3, which suggests that Spindlin1 is a downstream effector of H3K4me3 generated in heterochromatic regions. Collectively, our work calls attention to an intriguing function of Spindlin1 as a potent H3 "K4me3-K9me3/2" bivalent mark reader, thereby balancing gene expression and silencing in H3K9me3/2-enriched regions.

Methylation-sensitive Amplified Polymorphism as a Tool to Analyze Wild Potato Hybrids.

Methylation-Sensitive Amplification Polymorphism (MSAP) is a versatile marker for analyzing DNA methylation patterns in non-model species. The implementation of this technique does not require a reference genome and makes it possible to determine the methylation status of hundreds of anonymous loci distributed throughout the genome. In addition, the inheritance of specific methylation patterns can be studied. Here, we present a protocol for analyzing DNA methylation patterns through MSAP markers in potato interspecific hybrids and their parental genotypes.

Aberrant PDK4 Promoter Methylation Preceding Hyperglycemia in a Mouse Model.

Diabetic prevalence is at speedy increase globally. Previous studies stated that other than genetics, factors such as environment, lifestyle, and paternal-maternal condition play critical roles in diabetes through DNA methylation in specific areas of the genome. The purpose of this study is to investigate the methylation pattern of the PDK4 promoter in streptozotocin-induced diabetic mice until the 12th week of the observation. The methylation pattern in the blood samples was analyzed periodically, while the pattern in the muscle sample was only analyzed at the end of the experiment using the blood of the sacrificed animals. Three methylated CpG site 1, CpG site 6, and CpG site 7 were analyzed and quantified based on the band density using bisulfite treatment and methylation-specific polymerase chain reaction (PCR). The hyperglycemia period was developed at the 9th week of experiment. However, there was a significant increase of methylation, specifically on CpG site 6 started from week 6 to week 12. This peculiar methylation on CpG site 6 of PDK4 promoter in the blood sample before the hyperglycemic period might serve as a potential biomarker for early detection of diabetes in the patients. No significant difference was found between the methylation level of streptozotocin (STZ)-treated mice and of the control group in the muscle sample.